文献类型：期刊文献

中文题名：竹林金针虫实时荧光定量PCR内参基因的筛选与应用

英文题名：Screening and application of reference genes for qRT-PCR in bamboo wireworm

作者：叶碧欢[1] 陈友吾[1] 舒金平[2] 张威[2] 张亚波[2] 李海波[1] 宋其岩[1]

第一作者：叶碧欢

机构：[1]浙江省林业科学研究院,浙江杭州310023;[2]中国林业科学研究院亚热带林业研究所,浙江杭州311400

年份：2021

卷号：38

期号：3

起止页码：644-651

中文期刊名：浙江农林大学学报

外文期刊名：Journal of Zhejiang A & F University

收录：CSTPCD;;北大核心:【北大核心2020】；CSCD:【CSCD2021_2022】；

基金：浙江省自然科学基金资助项目(LQ19C160006);浙江省省属科研院所资助项目(2019F1068-1)。

语种：中文

中文关键词：森林保护学;竹林金针虫;平沙绿僵菌;内参基因;实时荧光定量PCR

外文关键词：forest protection;bamboo wireworm;Metarhizium pingshaense;reference gene;quantitative realtime PCR(qRT-PCR)

分类号：S763.38

摘要：【目的】筛选平沙绿僵菌Metarhizium pingshaense侵染条件下筛胸梳爪叩甲Melanotus cribricollis幼虫稳定表达的内参基因,为该竹林金针虫相关基因的表达研究奠定基础。【方法】基于筛胸梳爪叩甲幼虫的转录组数据,利用实时荧光定量PCR扩增特异性引物,分析相关性和扩增效率;利用GeNorm、NormFinder和BestKeeper软件评估筛选出6个候选内参基因(β-actin、GAPDH、α-tubulin、RPL13α、RPS3、RPS27a),并验证其表达稳定性。【结果】GeNorm分析结果显示:PRS27a和RPS3的表达最稳定,随后依次是α-tubulin、RPL13α、β-actin和GAPDH;最适合的内参基因数目为2。NormFinder分析结果显示:RPL13α的表达最稳定,随后依次是α-tubulin、RPS3、RPS27a、β-actin和GAPDH。BestKeeper分析结果显示:β-actin和GAPDH的P>0.5,不适合作为本试验条件下的内参基因。不同软件分析得出的候选内参排序存在一定差异。综合分析和表达稳定性验证表明PRS27a或RPS3是最佳内参基因,6个目的基因的表达水平变化趋势均基本一致。【结论】PRS27a和RPS3是研究平沙绿僵菌侵染的竹林金针虫相关基因表达的最佳内参基因。
[Objective] The objective of this study was to screen the stable expression of internal reference genes of Melanotus cribricollis larvae infected by Metarhizium pingshaense, so as to lay a foundation for the research on related gene expression in this bamboo wireworm. [Method] Based on the transcriptome data of M. cribricollis larvae, the correlation(R2) and amplification efficiency were analyzed by qRT-PCR with specific primers. The 6 candidate reference genes including β-actin, GAPDH, α-tubulin, RPL13α, RPS3 and RPS27 a were evaluated by GeNorm, NormFinder and BestKeeper softwares. The stabilities of selected candidate reference genes including PRS27 a and RPS3 were further validated by analyzing the expression of 6 target genes. [Result] GeNorm analysis showed that the expression of PRS27 a and RPS3 were the most stable,followed by α-tubulin, RPL13α, β-actin and GAPDH. The most suitable number of internal reference genes was2. NormFinder analysis showed that the expression of RPL13α was the most stable, followed by α-tubulin, RPS3, RPS27 a, β-actin, and GAPDH. BestKeeper analysis showed that the P values of β-actin and GAPDH were >0.5, which were not suitable for reference genes under the condition of this experiment. There were some differences in the ranking of candidate internal parameters obtained by different software analysis.Comprehensive analysis and expression stability verification showed that PRS27 a or RPS3 were the best internal reference genes, and the expression levels of 6 target genes were basically the same. [Conclusion]PRS27 a and RPS3 were the most appropriate reference genes for q RT-PCR analysis in bamboo wireworm infected by M. pingshaense.